<p>Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) represent a critical subset of immunosuppressive cells within the tumor microenvironment. Their interaction with breast cancer stem cells (BCSCs) constitutes a vital link that drives the malignant progression of breast cancer, particularly in triple-negative breast cancer (TNBC). However, the precise molecular mechanisms through which these cells mediate BCSC-related stemness reprogramming and enhanced metastatic potential in breast cancer remain inadequately understood. In this study, PMN-MDSCs were isolated from mouse breast cancer solid tumor tissues. Functional characterization was conducted using flow cytometric phenotyping, nuclear morphology analysis, and T cell immunosuppressive function assays. In vivo tumorigenesis experiments confirmed their pro-tumor growth effects and enhanced tumor stem cell characteristics. Concurrently, the CD44^hiCD24^lo subpopulation of 4T1 cells was sorted to systematically verify their tumor stem cell properties and stemness plasticity. In vitro co-culture experiments demonstrated that PMN-MDSCs significantly increased the proportion of CD44^hiCD24^lo stem-like subpopulations in 4T1 cells, prompting non-stem cells to differentiate into stem cell-like cells. They also promoted STAT3 phosphorylation, upregulated the expression of stemness-related proteins and mesenchymal markers, downregulated the epithelial marker E-cadherin, and markedly enhanced CXCL5 secretion. These effects were effectively reversed by the stemness inhibitor Napabucasin. Transwell non-contact co-culture and exosome function assays further confirmed that PMN-MDSCs regulate cancer cell stemness in a paracrine manner. S100A9, a key effector molecule in their exosomes, induced CXCL5 secretion by activating the STAT3 pathway. CXCL5 subsequently activated both the ERK1/2 and STAT3 pathways through autocrine and paracrine mechanisms, enhancing tumor cell metastatic potential and establishing a positive feedback loop that sustains its own secretion. This loop effect was blocked by a CXCL5-neutralizing antibody. Single-cell sequencing data analysis and clinical sample validation of TNBC indicated significantly elevated infiltration of myeloid cells, as well as increased S100A9 and CXCL5 expression levels in cancer tissues, alongside a marked upregulation of the mRNA stemness index. The stemness index was closely associated with poor patient prognosis. In summary, this study reveals that PMN-MDSCs can activate the STAT3-CXCL5-ERK positive feedback regulatory axis via exosomal S100A9, synergistically enhancing breast cancer cell stemness and metastatic capacity. These findings provide a theoretical reference and potential intervention targets for targeting the tumor microenvironment to inhibit TNBC progression.</p>